Communication cellulaire et switch moléculaire chez Bacillus cereus – Cell.com

The Cell.com project focuses on the study of a cell-cell communication system functioning in bacteria of the Bacillus cereus group (ie. B. anthracis, B. thuringiensis and B. cereus sensu stricto). This biological system functions according to a quorum-sensing mechanism involving a regulatory protein (NprR) and a signalling peptide (NprX). The originality of this system resides in the structural and functional properties of NprR, which appears to be a dual-function protein activated or inhibited by its cognate signaling peptide. When NprR is not bound to NprX, it negatively affects sporulation, but, once combined to NprX, it loses its initial activity and becomes a transcriptional activator. The close relationship existing between NprR and the Rap phosphatases suggests that the negative activity of NprR on sporulation is due to a phosphatase activity: NprR would desphosphorylate an intermediate of the sporulation phosphorelay, thus reducing the concentration of Spo0A~P and the sporulation rate. In presence of NprX, our results show that the couple NprR/NprX has no more effect on sporulation, but activates the transcription of nprA, a gene encoding a metalloprotease described as a virulence factor produced during sporulation. Consequently, NprX induces a functional shift allowing the bacteria (or a part of the bacterial population) to engage a new developmental program. We aim at analysing the different stages to this biological system, from the molecular interaction between NprR and NprX to the effects of this interaction on sporulation and nprA gene expression. Complementary work programs based on a multidisciplinary approach have been defined to achieve our objectives. A part of the project focuses on the NprR/NprX structure-function and on the two opposite effects generated by this protein-peptide interaction. We have demonstrated that the NprR/NprX interaction activates the transcriptional regulator function of NprR and, conversely, inhibits its initial function (the presumed Rap-like activity). Our project is to determine how the binding of NprX can switch the function of the protein. Data obtained on other Gram-positive quorum-sensing systems suggest that NprX switches on the transcriptional activator function of NprR by rearranging the HTH domains into an appropriate DNA-binding conformation. However, the conformational change resulting in the loss of the phosphatase activity remains obscure, since the 3D structure of the Rap phosphatases is unknown. The understanding of the structural changes shifting the activity of NprR should provide new highly significant insights into the regulatory mechanism of this bifunctionnal protein. Another part of the project focuses on the functioning and on the role of the NprR/NprX quorum-sensing system. We aim to determine how bacteria are able to integrate multiple signalling pathways to adapt their behaviour to both bacterial density and environmental conditions. This section includes four interdependent points: (i) The characterization of the regulatory circuits converging toward the formation of the NprR/NprX complex acting as a transcriptional activator. This includes the study of nprR and nprX expression and the study of the NprX export-import system. (ii) The identification of biologically relevant substrates of NprR. The role of these substrates on sporulation and the role of NprR on the substrates will be studied. (iii) The role of NprR/NprX as a transcriptional activator. The regulation of nprA expression by NprR/NprX will be specifically analysed, and the complete list of NprR/NprX-regulated genes will be determined by a transcriptomic analysis. (iv) The study of bistability in B. cereus. Due to the major role of the phosphorelay as a source of bistabilty in B. subtilis, a close attention will be paid to the bistability states generated by NprR and NprR/NprX in stationary phase cultures of B. cereus. To summarize: the project is to associate multidiscplinary approaches to study a regulator displaying two distinct functions. The main orginality of this project is the molecular switch responsible for the dual function of the NprR protein. Moreover, this exceptional molecular property is a remarkable tool to study stochastic situations prevailing during stationary phase and preceding the ultimate and not reversible decision to sporulate. NprR/NprX provides an excellent model for studying how a single protein complex integrates two functions able to coordinate gene expression and development.